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Translation of abstract (English)

DNA contains the genetic instructions used for the development and function of all living organisms. In eukaryotes, a large amount of DNA must be compacted into the volume of nucleus; this is accomplished by wrapping short fragments of DNA around a core of histone proteins forming a complex called nucleosome. These nucleosomes, however, represent an obstacle for the cellular machinery that reads the genetic information. To regulate which information is read out at any given time, the cell can assemble or disassemble specific nucleosomes at the loci of interest. In this thesis, an in vitro assay for the investigation of these regulatory processes has been developed. For the assay, nucleosomes were fluorescently labeled on different positions within the histone core, as well as along the DNA. They were then analyzed by confocal microscopy using fluorescence correlation spectroscopy (FCS) and single pair Förster Resonance Energy Transfer (spFRET) while assembly and disassembly was induced by varying the ionic strength of the medium. FCS can be used to measure the diffusion coefficient by analyzing fluctuations in fluorescence emission as the molecules move through the confocal spot. As the diffusion coefficient is a measure for the molecule’s size and shape, distinct (dis)assembly steps could be observed using FCS showing that the histone core disassembles sequentially and sub-stoichometric histone-DNA complexes arise where the DNA becomes more accessible to the cellular machinery. While step-wise (dis)assembly proved to be an interesting finding, intermediate states within this pathway were further probed using spFRET. FRET is the non radiative energy transfer between two fluorophores. Its efficiency depends highly on the fluorophores’ distance which can thus be investigated. Compared to bulk analysis, spFRET experiments can unravel specific subpopulations of heterogeneous samples that differ in distance between their fluorescently labeled components. In this thesis a previously uncharacterized structural state was observed, in which the histones become more accessible to the cellular machinery prior to dissociation from the DNA. This intermediate state is populated at 0.2 – 6 % under physiological conditions. In vivo, the stability of nucleosomes is regulated by exchanging major type histones for histone variants. To characterize the role of histone variants in nucleosome accessibility, major-type H2A was replaced with histone variant H2A.Z in nucleosomes, and its effect on nucleosome (dis)assembly and open-state formation was investigated. Strikingly, spFRET data analysis shows nucleosomes containing H2A.Z follow a (dis)assembly pathway distinct from nucleosomes containing major-type histones, which also contains new, yet to be identified, intermediate states.